Infectious disease is a global threat to human health. The World Health Organization notes a pressing need to develop novel antimicrobial strategies that limit the impact of these life-threatening pathogens. These pathogens include the major causative agents of nosocomial infections, e.g., Staphylococcus aureus, and a major respiratory pathogen responsible for community-acquired pneumonia and morbidity world-wide, Streptococcus pneumoniae. Each is becoming increasingly multidrug-resistant severely complicating treatment options. In this proposal, we seek to integrate our fundamental studies of bacterial transition metal (manganese, copper and zinc) homeostasis, sulfur metabolism and sulfide homeostasis to accelerate the pace of discovery of novel antibacterial strategies. We have long-standing interests in the transcriptional repressors and more recently, metal trafficking proteins, that allow a bacterium to adapt to host-mediated remodeling of transition metal availability. We've discovered and structurally characterized new players in this process in M. tuberculosis, S. aureus and S. pneumoniae and have framed our quantitative investigations of these systems as allosteric inorganic switches that orchestrate metal homeostasis and resistance to toxicity in cells. These studies led directly to the discovery and ongoing elucidatio of what we anticipate represents a novel, highly specific regulatory response to reactive sulfur species (RSS) and potentially, reactive nitrogen oxide species (nitroxyl; HNO) in S. aureus. We hypothesize that this response impacts the ability of S. aureus and other pathogens to regulate colonization and nitric oxide (NO)-mediated dispersal of biofilms (biofilm dynamics) and resistance to antibiotic-induced oxidative stress. Future studies will be carried out in three general areas: 1) biological characterization and structural/dynamics studies, using state-of-the-art methyl-specific NMR relaxation experiments, of new allosteric systems involved in metalloregulation of transcription and regulation of RSS and RNOS; 2) obtaining new molecular-level insights into copper resistance and manganese homeostasis in S. pneumoniae, and mechanisms of adaptation to extreme zinc limitation induced by host-mediated nutritional immunity in Acinetobacter baumannii, and 3) holistically probe the cellular response to sulfide and RNOS stress using transcriptomic, mass spectrometry-based profiling of proteome cysteine thiol oxidative modifications, and targeted metabolite profiling approaches, with the goal to identity new players and mechanisms in this process. Our multidisciplinary approach, which seamlessly spans biophysical chemistry to microbial physiology, enhances the probability of transforming our understanding of fundamental features of transition metal homeostasis linked to virulence and a completely unexplored cellular response to RSS/RNOS in important human pathogens.